In plants, multiple lineages have evolved sex chromosomes independently, providing a powerful comparative framework, but few specific determinants controlling the expression of a specific sex have been identified. We investigated sex determinants in the Caucasian persimmon, Diospyros lotus, a dioecious plant with heterogametic males (XY). Male-specific short nucleotide sequences were used to define a male-determining region. A combination of transcriptomics and evolutionary approaches detected a Y-specific sex-determinant candidate, OGI, that displays male-specific conservation among Diospyros species. OGI encodes a small RNA targeting the autosomal MeGI gene, a homeodomain transcription factor regulating anther fertility in a dosage-dependent fashion. This identification of a feminizing gene suppressed by a Y-chromosome-encoded small RNA contributes to our understanding of the evolution of sex chromosome systems in higher plants.

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Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of the plant and also to the taste of the fruit, mainly through astringency. Persimmon (Diospyros kaki) is unique in being able to accumulate abundant PAs in the fruit flesh. Fruits of the nonastringent (NA)-type mutants lose their ability to produce PA at an early stage of fruit development, while those of the normal astringent (A) type remain rich in PA until fully ripened. The expression of many PA pathway genes was coincidentally terminated in the NA type at an early stage of fruit development. The five genes encoding the Myb transcription factor were isolated from an A-type cultivar (Kuramitsu). One of them, DkMyb4, showed an expression pattern synchronous to that of the PA pathway genes in A-and NA-type fruit flesh. The ectopic expression of DkMyb4 in kiwifruit (Actinidia deliciosa) induced PA biosynthesis but not anthocyanin biosynthesis. The suppression of DkMyb4 in persimmon calluses caused a substantial down-regulation of the PA pathway genes and PA biosynthesis. Furthermore, analysis of the DNA-binding ability of DkMyb4 showed that it directly binds to the MYBCORE cis-motif in the promoters of the some PA pathway genes. All our results indicate that DkMyb4 acts as a regulator of PA biosynthesis in persimmon and, therefore, suggest that the reduction in the DkMyb4 expression causes the NA-type-specific down-regulation of PA biosynthesis and resultant NA trait.

Dioecy, the presence of male and female flowers on distinct individuals, has evolved independently in multiple plant lineages, and the genes involved in this differential development are just starting to be uncovered in a few species. Here, we used genomic approaches to investigate this pathway in kiwifruits (genus ). Genome-wide cataloging of male-specific subsequences, combined with transcriptome analysis, led to the identification of a type-C cytokinin response regulator as a potential sex determinant gene in this genus. Functional transgenic analyses in two model systems, and , indicated that this gene acts as a dominant suppressor of carpel development, prompting us to name it (). Evolutionary analyses in a panel of species revealed that is located in the Y-specific region of the genome and probably arose from a lineage-specific gene duplication. Comparisons with the duplicated autosomal counterpart, and with orthologs from other angiosperms, suggest that the -specific duplication and subsequent evolution of-elements may have played a key role in the acquisition of separate sexes in this species.

Epigenetic regulation can add a flexible layer to genetic variation, potentially enabling long-term but reversible cis-regulatory changes to an allele while maintaining its DNA sequence. Here, we present a case in which alternative epigenetic states lead to reversible sex determination in the hexaploid persimmon Diospyros kaki. Previously, we elucidated the molecular mechanism of sex determination in diploid persimmon and demonstrated the action of a Y-encoded sex determinant pseudogene called OGI, which produces small RNAs targeting the autosomal gene MeGI, resulting in separate male and female individuals (dioecy). We contrast these findings with the discovery, in hexaploid persimmon, of an additional layer of regulation in the form of DNA methylation of the MeGI promoter associated with the production of both male and female flowers in genetically male trees. Consistent with this model, developing male buds exhibited higher methylation levels across the MeGI promoter than developing female flowers from either monoecious or female trees. Additionally, a DNA methylation inhibitor induced developing male buds to form feminized flowers. Concurrently, in Y-chromosome-carrying trees, the expression of OGI is silenced by the presence of a SINE (short interspersed nuclear element)-like insertion in the OGI promoter. Our findings provide an example of an adaptive scenario involving epigenetic plasticity.

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3'5' hydroxylase (F3'5'H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3' hydroxylation (F3'H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3'5'H to F3'H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3'5'H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.

MethodsMethod S1: Screening of the expressed candidate sex determinants Developing anthers at stage 1-2, which correspond to the differentiation stage of male or female androecium (see Supplementary Figure S1), were sampled from F1 sibling vines derived from an interspecific cross, A. rufa sel. Fuchu × A. chinensis sel. FCM1, named KE population (15), planted on Kagawa University, Japan (N34.28, E134.13), in 10-22 April in 2016-2017. Total RNA was extracted using the Plant RNA Reagent (Invitrogen) and purified by phenol/chloroform extraction. Two micrograms of total RNA were processed in preparation for Illumina Sequencing, according to a previous report (15). The constructed libraries were sequenced on Illumina's HiSeq 4000 sequencer (50-bp single-end or 150-bp paired-end reads). All Illumina sequencing were conducted at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, and the raw sequencing reads were processed using custom Python scripts developed in the Comai laboratory and available online (http://comailab.genomecenter.ucdavis.edu/index.php/), as previously described (9). Male-specific Ychromosomal sequences in kiwifruit, defined MSY contigs, were comprehensively identified in previous study (15). The mRNA-Seq reads from each 5 male and female individuals from the KE population (Supplemental Table S11) (15) were used to identify the genes substantially expressed in developing anthers. The mRNA-Seq reads were aligned to the hypothetical 61 genes located on the 249 MSY contigs (Akagi et al. 2018), using the Burrows-Wheeler Aligner (BWA) (37) allowing up to ca 3% mismatches. The number of reads mapping to each contigs was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). For Friendly Boy (FrBy), which showed male-specific and anther-enriched expression, the expression patterns were further examined using various plant organs and developing anthers (stage 2a, 2b, 3a, and 3b, see Supplementary Figure S1). Method S2: Expression profiling in kiwifruit antherThe described mRNA-Seq reads from each 5 male and female individuals of the KE population were aligned to the whole CDS sequences sets in A. chinensis (27), using BWA with default parameters. The number of reads mapped to each reference sequences was recorded from the alignment file produced by the Sequence Alignment/Map (SAM) tool (38) (http://samtools.sourceforge.net/). The read counts per gene were generated from the aligned SAM files using a custom R script. Differential expression between male and female individuals was analysed in R (version 3.0.1) using the R package DESeq (Anders and Huber, 2010) (version 1.14; http://bioconductor.org/packages/release/bioc/html/DESeq.html). We conducted DESeq analysis using 5 biological replicates from male and female individuals, with the following parameters: method='per-condition' and sharingMode='maximum'. An FDR threshold of 0.1 was used to identify differentially expressed genes. Method S3: in situ RNA hybridizationRNA in ...

Dioecy, the presence of male and female flowers on separate individuals, is both widespread and uncommon within flowering plants, with only a few percent of dioecious species spread across most major phylogenetic taxa. It is therefore safe to assume that dioecy evolved independently in these different groups, which allows us to ask questions regarding the molecular and developmental mechanisms underlying these independent transitions to dioecy. We start this review by examining the problem from the standpoint of a genetic engineer trying to develop dioecy, discuss various potential solutions, and compare them to models proposed in the past and based on genetic and evolutionary considerations. Next, we present recent information regarding candidate sex determinants in three species, acquired using newly established genomic approaches. Although such specific information is still scarce, it is slowly becoming apparent that various genes or pathways can be altered to evolve dioecy.